Abstract
DNA-O6-methylguanine methyltransferase was purified from the nuclear fraction of fresh human placenta using ammonium sulphate precipitation, gel filtration, affinity chromatography on DNA-cellulose and hydroxyapatite. The methyltransferase preparation was approximately 1–2% pure based on specific activity, and was free of nucleic acids. The protein reacts stoichiometrically with O6-methylguanine in DNA with apparent second-order kinetics. The human methyltransferase has a pH optimum of about 8.5, similar to that of the corresponding rat and mouse proteins. NaCl inhibits the reaction in a concentration-dependent fashion. The human protein, like the rodent andE. coli methyltransferases, needs no cofactor. While lmM MnCl2, lmM spermidine, 5mM MgCl2 and 10 mM EDTA individually do not significantly inhibit the initial rate of reaction, the protein is nearly completely inactive in 5 mM A1Cl3 or FeCl2 or 10 mM spermidine. The initial rate of reaction increases as a function of temperature at least up to 42°. The reaction is inhibited by DNA in a concentration-dependent manner, with single-stranded DNA being more inhibitory than duplex DNA.
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Abbreviations
- EDTA:
-
ethylenediaminetetraacetic acid
- DTT:
-
dithiothreitol
- αToSF:
-
α-toluenesulphonyl fluoride
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Boulden, A.M., Foote, R.S., Fleming, G.S. et al. Purification and some properties of human DNA-O6-methylguanine methyltransferase. J. Biosci. 11, 215–224 (1987). https://doi.org/10.1007/BF02704671
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DOI: https://doi.org/10.1007/BF02704671